Chapter 2

Plant Electrostimulation
and Data Acquisition

Emil Jovanov and Alexander G. Volkov

Abstract Plant electrostimulation is a very efficient method for evaluation of

biologically closed electrical circuits in plants. The information gained from plant
electrostimulation can be used to elucidate and observe the intracellular and
intercellular communication in the form of electrical signals within plants.
Monitoring the electrical signaling in higher plants represents a promising method
to investigate fast electrical communication during environmental changes. Here
we discuss DC methods of plant electrostimulation and describe a new Charge
Stimulation Method in plant electrophysiology. It is often convenient to represent
the real electrical and electrochemical properties of biointerfaces with idealized
equivalent electrical circuit models consisting of discrete electrical components.
Biologically closed electrical circuits in plants can be investigated using the
Charge Stimulation Method.

2.1 Introduction

The electrical phenomena in plants have attracted researchers since the eighteenth
century (Bertholon 1783; Bose 1907, 1913, 1918, 1926, 1928; Burdon-Sanderson
1873; Davies 2006; Keller 1930; Ksenzhek and Volkov 1998; Lemström 1904;

E. Jovanov (&)
Electrical and Computer Engineering Department,
University of Alabama in Huntsville, Huntsville, AL 35899, USA
e-mail: emil.jovanov@uah.edu
A. G. Volkov
Department of Chemistry, Oakwood University,
7000 Adventist Blvd, Huntsville, AL 35896, USA
e-mail: agvolkov@yahoo.com

A. G. Volkov (ed.), Plant Electrophysiology, 45

DOI: 10.1007/978-3-642-29119-7_2,  Springer-Verlag Berlin Heidelberg 2012
46 E. Jovanov and A. G. Volkov

Sinukhin and Britikov 1967; Volkov 2006a, b). Biologically closed electrical
circuits (Nordestrom 1983) operate over large distances in biological tissues. The
activation of such electrical circuits can lead to various physiological and
biochemical responses. The cells of many biological organs generate electric
potentials that can result in the flow of electric currents (Volkov et al. 1998).
Electrical impulses may arise as a result of stimulation. Once initiated, these
impulses can propagate to adjacent excitable cells. The change in transmembrane
potential can create a wave of depolarization which affects the adjoining, resting
membranes. Thus, while the plasma membrane is stimulated at any point, the
action potential can propagate over the entire length of the cell membrane and
along the conductive bundles of tissue with constant amplitude, duration, and
speed (Volkov 2006b). Characteristic length of action potentials is defined as the
propagation speed multiplied by the duration of the action potential. To detect the
real action potentials, the distance between electrodes should exceed the charac-
teristic length of an action potential. Graded, electrotonic, and variation potentials
propagate with decreasing amplitude. Electrical signals can propagate along the
plasma membrane on short distances in plasmodesmata, and on long distances in
conductive bundles. Action potentials in higher plants hold promise as the infor-
mation carriers in intracellular and intercellular communication during environ-
mental changes (Volkov 2000, 2006b).
Measurement of plant electrical activity and evoked potentials raise a number
of challenging issues, including type and position of electrodes, reference poten-
tials, methodology of measurement, and synchronization with external events.
Omissions and mistakes in methodology may lead to incorrect conclusions about
the nature of underlying processes. One of the typical mistakes is creating direct
analogies between standard electrical circuits and electrical circuits in plants.
Electrical circuits have clearly defined reference potential (‘‘common ground’’)
and all potentials are measured relative to the ground potential. Most potentials in
computer systems are digital and exhibit high immunity to noise. In contrast,
plants exhibit a hierarchical structure with no common ground potential, many
signals are nonlinear, and have poor signal to noise ratio.
Scientific hypotheses could be tested using electrical stimulation of plants and
monitoring of biological effects caused by the stimulation.
In this chapter we present methods for monitoring and stimulation of plant’s
electrical activity. In addition to fundamental theoretical concepts we present our
experience in the configuration and development of the custom data acquisition
and plant DC stimulation systems, and results from plant experiments.

2.2 Data Acquisition

Although many processes in plants are slow enough for direct observation, a number
of processes are too quick and require additional instrumentation for scientific study.
Typical example is mechanical closing of carnivorous plants, such as the Venus
2 Plant Electrostimulation and Data Acquisition 47

Fig. 2.1 Block diagram of the data acquisition system

flytrap, that can close lobes and capture small insects in a fraction of the second
(Markin et al. 2008; Volkov et al. 2007, 2008a, b). In addition to fast cameras, it is
necessary to monitor electrical activity and plant signaling during closing.
Data acquisition is the process of converting analog physical signals into digital
numeric values that can be stored, processed, and visualized by a computer. Data
acquisition systems are often represented by the acronyms DAS or DAQ. Recent
development of embedded computer systems and standard data acquisition boards
lead to the development of virtual instrumentation that allows use of common
hardware with custom software to represent virtual instruments for a variety of
applications.
Typical components of data acquisition systems include:
• Sensors that convert physical parameters to electrical signals that can be
processed by the data acquisition system,
• Signal conditioning circuits to convert sensor signals into a form that can be
converted to digital values,
• Analog-to-digital converters, which convert conditioned sensor signals to digital
values,
• Microprocessor-based controller that performs the following functions:
– user interface and control,
– file access and storage,
– networking for distributed systems,
– signal processing and analysis, and
– result presentation and visualization
For example, a digital thermometer might use thermistor as sensor to convert
temperature to variable resistance; signal conditioning circuit (such as voltage
divider or amplifier) to convert variable resistance to variable voltage, amplify and
filter signal; analog-to-digital converter can be used to convert the voltage to
digital values that are read and processed by the microcontroller, and displayed to
the user.
Analog-to-digital conversion assumes analog voltages relative to the reference
voltage.
Data acquisition applications are typically controlled by application-specific
programs that provide custom user-interfaces, processing, and presentation of
results (Fig. 2.1).
48 E. Jovanov and A. G. Volkov

0.04 0.04

0.02 0.02

0.00 0.00
E (V)

E (V)
-0.02 -0.02

-0.04 -0.04

-0.06 -0.06
-0.08 -0.08

0.000 0.002 0.004 0.006 0.008 0.010 0.000 0.002 0.004 0.006 0.008 0.010
Time (s) Time (s)
(a) 300,000 Samples/s (b) 100,000Samples/s

0.04 0.04
0.02 0.02
0.00 0.00
E (V)

E (V)
-0.02 -0.02
-0.04 -0.04
-0.06 -0.06
-0.08 -0.08

0.000 0.005 0.010 0.015 0.020 0 50 100 150 200

Time (s) Time (s)
(c) 1000 Samples/s (d) 10 Samples/s

Fig. 2.2 Reconstructed 500 Hz sinusoidal signal from the digitized signal sampled at a 300,000
samples/second, b 100,000 samples/second, c the Nyquist rate of 1,000 samples/second; d aliased
500 Hz signal due to under sampling at 10 samples/second

2.2.1 Sampling

Sampling represents the process of converting continuous analog signals with

unlimited time and amplitude resolution to discrete samples equivalent to the
instantaneous value of the continuous signal at the desired time points. Typical
sampling of the analog signal is represented in Fig. 2.2. Sampling involves time
and amplitude discretization, as described in the following sections.

2.2.1.1 Time Discretization

Continuous analog signal is converted to a sequence of discrete samples in discrete

time points that could be uniform or variable. Uniform sampling is commonly used,
where the sampling interval Ts determines sampling frequency or sampling rate Fs:
1
Fs ¼ ð2:1Þ
Ts
Sampling frequency determines the number of samples obtained in one second,
represented in samples per second or expressed in Hertz (Hz). For example,
2 Plant Electrostimulation and Data Acquisition 49

sampling frequency Fs = 100 Hz means that we will collect 100 samples of the
signal per second.
Fundamental limitation of sampling is represented by Nyquist–Shannon sam-
pling theorem (Shannon 1949) which shows that a sampled analog signal can be
perfectly reconstructed from an infinite sequence of samples if the sampling rate
exceeds 2  Fmax samples per second, where Fmax represents the highest frequency
of the original signal, also known as Nyquist frequency:
Fs  2 Fmax ð2:2Þ
Therefore, data acquisition systems must satisfy two conditions:
• The signal conditioning circuit must limit maximum frequency of the signal to
Fs max [Hz]; typically, this is implemented as a low pass or band pass filter with
maximum cutoff frequency of Fs max [Hz]. Please note that even without peri-
odic high-frequency components, fast changing signals have wide spectrum
(theoretically infinite spectrum) that must be limited for correct data acquisition.
• The data acquisition card must sample signals with sampling rate of at least
2  Fmax [Hz]. However, lower cutoff frequency of the low pass filter may distort
the signal in the presence of fast changing signals (see previous condition);
hence, cutoff frequency of the low pass filter is usually selected close to Fs/2.
Consequently:
Sampling frequency Fs is selected to preserve most of the frequency con-
tent and shape of the signal, and data acquisition systems must use low pass
filter with cutoff frequency not higher than Fs/2.

Figure 2.3 represents an example of inadequate sampling frequency and wrong

conclusions that might be drawn from the measurement. A sequence of fast regular
pulses sampled at low frequency might generate the impression that the underlying
phenomenon is a single, slow changing pulse, as represented in the lower plot of
Fig. 2.3.
General purpose voltmeters typically represent slow data acquisition systems
with sampling rate in the order of few samples per second. Therefore, some high
speed changes might generate false impression of the underlying phenomena, as
represented in Fig. 2.3.

2.2.1.2 Quantization and Coding

Analog samples are converted to digital values using Analog-to-Digital or AD

Converters. AD converter represents a quantizer with a number of discrete levels
against which the sampled amplitude is compared to generate a binary code
representing amplitude of the current sample. The number of levels is defined by
the resolution or number of bits (nb) of the AD converter. The value of the
50 E. Jovanov and A. G. Volkov

Fig. 2.3 Illustration of data acquisition with inadequate sampling frequency; upper plot
synthetic signal as a sequence of pulses; lower plot reconstructed signal sampled with low
sampling frequency

quantization step D depends on the range and resolution of the AD converter and
can be represented as:
Vþ  V
D¼ ð2:3Þ
2nb
where V+ and V- represent positive and negative reference voltages, and
Vrange = V+ - V-. For example, a 12-bit AD converter with V+ = 5 V and
V- = 0 V has quantization step of:
5V  0V 5V
D¼ ¼ ¼ 1:22 mV ð2:4Þ
212 4096
Error generated by the quantization can be represented as a noise generated by
conversion. For the truncation quantizer the maximum error can be represented as:
Vrange
0eD ¼ ð2:5Þ
2nb
Therefore, signal to noise ratio and maximum noise can be controlled by the
resolution of the AD converter. Quantization and coding are represented in
Fig. 2.4.
If a single AD converter is used for multiple signals (multichannel configura-
tion), individual channels might use separate references (differential input) or a
single reference for all channels (single ended). Noise immunity is much better
with differential input, while single ended recording allows two times more
channels in the same data acquisition setup.
2 Plant Electrostimulation and Data Acquisition 51

AD converter code
1..11

1..10
…
0..11

0..10

0..01

0..00
input voltage [V]

V- V-+Δ V-+2Δ V-+3Δ V+

Vrange

Fig. 2.4 Quantization and coding

2.2.2 Signal Conditioning

Monitoring of electrical activity of plants creates several unique challenges:

• plant’s electrical activity generates very small voltages, typically in the order of
mV or tens of mV
• sources of plant’s electrical activity are very weak and must be amplified to
improve noise immunity of the signals
– amplifiers also need a reference point (referenced as ‘‘GROUND’’ in typical
electrical systems); however, choice of the reference point may significantly
influence signal generation and signal quality. The most convenient reference
point is soil around the plant; however, in many applications this configura-
tion is inadequate due to large resistance between sources of plant activity and
root/soil around the plant.
– separate source ground and measurement system grounds create difference in
ground potentials and ground loops, visible mostly as power line interference
(50 Hz in Europe or 60 Hz in America).
• long wires typically require differential acquisition or optical isolation of
sources of plant activity.

2.2.3 Impedance Matching

Sources of plant electrical activity can be represented as ideal voltage source with
series resistance, as represented in Fig. 2.5. Measured voltage (Vm) will depend on
the resistance of electrodes (Re), as well as input resistance of the measurement
device (Rin) and can be represented as:
52 E. Jovanov and A. G. Volkov

Fig. 2.5 Measured potential as a function of the internal resistance of the measurement
equipment; Re—electrode impedance, Rs—source impedance, Rin—input impedance of the
measurement system

Rin 1
Vm ¼ Vs ¼ Vs ð2:6Þ
Rin þ Rs þ 2Re 1 þ RRs þ 2R
in R
e
in

For very large values of the input resistance ðRin ! 1Þ; Vm  Vs :

Typical values for Re are in the order of a few kX for Ag/AgCl electrodes and
tens of MX for ion selective electrodes with membranes, while Rs is often in the
order of hundreds or thousands of kX. Therefore, input resistance of the data
acquisition system must be at least in the order of GX to accurately represent the
signal. That is the reason why low input resistance oscilloscopes cannot be used
without signal conditioning and amplification of the signal.

2.3 DC Methods of Electrostimulation

There are a few ‘‘methods’’ of plant electrostimulation such as using DC source of

voltage or electrical current (Houwink 1935, 1938; Jonas 1970; Mizuguchi et al.
1994; Volkov et al. 2007), function generator (Volkov et al. 2012), charge stim-
ulation method (Volkov et al. 2008a, b, c, 2009a, b, 2010a, b, c, d, 2011a, b, c, d)
or AC method of electrical impedance (Inaba et al. 1995; Laarabi et al. 2005;
Wang et al. 1994; Zhang and Willison 1991).

2.3.1 Function Generator

Function generators are routinely used for stimulation of the general purpose
electrical circuits. The function generator gives many options for the electrosti-
mulation: shapes, duration, frequency of stimulation, and offset voltage Uoffset, but
it cannot regulate the electrical charge or electrical current during the plant
electrostimulation.
2 Plant Electrostimulation and Data Acquisition 53

Function generator actively drives all states of the generated signal. For
example, for a square wave signal, function generator will generate inactive states
as voltage equal to 0 V and active state at a certain voltage (e.g., 1 V output). This
means that the function generator connected to the plant will actively force 0 V
voltage even during inactive state that will in most cases interfere with the resting
state of the plant (approximately 20–60 mV generated by the plant). Similarly,
when a function generator is used to apply pulses with a given potential, it
generates a potential of zero volts when the pulse is not being applied. For
example, when the generator applies a 100 mV pulse lasting for 1 s, the function
generator will generate a 0 mV output before and after the pulse. This means that
the native plant potential will be forced to zero during periods of no stimulation,
which interferes with the normal plant signaling mechanism and electrical
recovery of the plant.
Therefore, stimulation using function generator interferes with the electrical
recovery of the plant. That is the reason why we used charge stimulation method.

2.3.2 Charge Stimulation Method

Charged capacitor applies electrical potential between two electrodes decreasing

gradually to zero during the discharge of a capacitor. A function generator
maintains a given high or low potential in the electrodes. For this reason, the
electrical response during the potential increase or decrease from a function
generator has corresponding positive or negative amplitude. Therefore, in order to
estimate the plant’s response after stimulation, we have to effectively ‘‘discon-
nect’’ the function generator from the plant and monitor the plant’s electrical
response. This feature is not available with standard function generators.
We propose the use of the charge stimulation method that allows delivery of an
electrical charge and disconnection from the plant. The charge is delivered from a
capacitor that is charged at a given potential. When a capacitor with capacitance
C is connected to the source with potential voltage U, the total capacitor charge is
Q = CU, which allows precise regulation of the amount of charge during stimu-
lation by using different capacitors and applying various voltages. A mechanical or
electronic switch can instantaneously connect the charged capacitor to the plant
and induce a response for a given stimulation period and disconnect the capacitor
to monitor the plant’s response (Volkov et al. 2010b, 2011a).
We applied a novel electrostimulation method, as presented in Fig. 2.6, to allow
separate control of both amplitude and timing of the stimulation pulse and to
provide a high-impedance optical isolation when the plant is not stimulated. We
used a custom board with microcontroller Texas Instruments MSP430F149 to
generate logic pulse of the precisely controlled duration on user’s request. The
pulse triggers a signal conditioning circuit with preset reference voltage through
the optocoupler. This approach effectively disconnects the plant from the stimu-
lation system when the pulse is not present. The amplifier allows active driving of
54 E. Jovanov and A. G. Volkov

LIGHT

FARADAY
CAGE Data Acquisition System

Battery

+ Vref
-

Microcontroller User
pulse generator input
Optocoupler

Fig. 2.6 Experimental setup for direct current electrostimulation of a plant with optical isolation

the pulse that results in much faster recharging of the capacitor. A separate battery
was used to eliminate high-frequency interference of the pulse generator system.
Using our new DC electrostimulation system, it was evident that the application
of an electrical stimulus between the midrib (positive potential) and a lobe (neg-
ative potential) causes the Venus flytrap to close the trap without any mechanical
stimulation in 0.3 s after electrical stimulation (Volkov et al. 2007). The average
stimulation pulse voltage sufficient for rapid closure of the Venus flytrap was
1.50 V (standard deviation is 0.01 V, n = 50) for 1 s. The inverted polarity pulse
with negative voltage applied to the midrib could not close the plant. We were
unable to open the plant by applying impulses in the same voltage range with
different polarities for pulses of up to 100 s.
The primary objective of our experiments was to precisely determine the con-
ditions of the charged electrical stimulation that generate a given biological effect.
We implemented two types of electrostimulation: a manual switch and a custom
made specific controller. Manual stimulation is convenient for single stimulation
because it does not require additional equipment. It was implemented using a
double pole double throw (DPDT) switch to connect the known capacitor to the
voltage source during charging and then to the plant during plant stimulation to
induce a response. However, manual switching does not allow precise control of
timing of the stimulation. Therefore, we designed and implemented a custom plant
stimulator to allow multiple stimulations with precise control of timing and voltage
during stimulation. The plant stimulator is a battery-powered portable device
controlled by a low-power microcontroller, MSP430F1611 (Texas Instruments,
Texas, USA). A specialized PC program allows flexible configuration of the con-
troller and communicates with the controller through optically isolated USB
interface. During each stimulation cycle, the controller charges capacitor with
2 Plant Electrostimulation and Data Acquisition 55

predefined voltage using integrated digital to analog (DA) converter of the mi-
crocontroller. A dual integrated analog switch controlled by the microcontroller
connects the capacitor to DA converter during charging and to the plant during
stimulation, allowing stimulation with microsecond resolution.
Each pulse can be controlled with resolution of 30 ls. The stimulation voltage
can be set in steps of 0.6 mV to maximum voltage of 2.5 V. After charging, the
capacitor with capacitance C charged with voltage Vs contains the amount of
charge equal to Q1 = C Vs if the voltage after stimulation falls to Vps the
remaining charge will be Q2 ¼ C Vps : Therefore, the stimulation delivered charge
DQ ¼ Q1  Q2 ¼ CðVs  Vps Þ: A dual integrated SPDT analog switch is
controlled by the microcontroller and connects the capacitor to DA converter
during charging and to the plant during stimulation. After N stimulations, the total
P
amount of charge delivered is equal to Qs ¼ C  Ni¼1 ðVi  Vpi Þ: The proposed
approach can therefore precisely define stimulation timing and charge. For a given
capacitance of the stimulation capacitor, the user can select the number of
stimulations N and the stimulation period.
For each stimulation, the capacitor was connected to electrodes in the plant
until complete discharge and then disconnected from the electrodes. The following
experiments with different conditions were performed at least 5 min later,
although there was no noticeable difference in response for different time periods
between stimulations. Voltage in the plant was measured between experiments, but
no additional effects were detected between experiments. Electrodes remained in
the plant between experiments.
It is possible to estimate charge Q = UC, electrical current I = -dQ/dt, work
W = U2C/2, power P = UI, and resistance R = U/I from the time dependence of
a capacitor discharge in a plant tissue as it is shown in Fig. 2.7 for a capacitor
discharge in the Venus flytrap.
The charge stimulation method (Volkov et al. 2008a, b, 2009a, b, 2010a, b, c, d)
was used to estimate, with high precision, the amount of electrical energy nec-
essary to induce a response.

2.3.2.1 Mathematical Treatment of a Capacitor Discharge in Passive

Electrical Circuits in Plants Developed by Professor V. S. Markin

If a capacitor of capacitance C with initial voltage U0 is discharged during time

t through a resistor R (Fig. 2.8a), the voltage across the capacitor decreases
exponentially with time t:

UðtÞ ¼ U0  et=s ð2:7Þ

where s = RC denotes the time constant and U0 is the initial voltage of a capacitor
(Feynman et al. 1963). Equation 2.7 in logarithmic form reads:
log10 UðtÞ ¼ log10 U0  t=2:3 ð2:8Þ
56 E. Jovanov and A. G. Volkov

(a) (c)
1.4 1.5
1.2
1.2
1.0
U/V

0.9

I /μA
0.8
0.6 0.6
0.4
0.2 0.3

0.0 0.0
0 2 4 6 8 10 0 2 4 6 8 10
Time / s Time / s
(b) (d) 5
6
4

W /μ J
Q /μ C

4
2
2
1

0 0
0 2 4 6 8 10 0 2 4 6 8 10
Time / s Time / s

Fig. 2.7 Time dependencies of voltage U a charge Q b electrical current I c, and electrical
energy W d during electrostimulation of the Venus flytrap upper leaf by 4.7 lF charged capacitor
with initial voltage U0 of 1.5

The time constant s can be determined from the slope of this linear function.
At s = RC, the capacitor charge is reduced to CU0e-1, which is about 37% of its
initial charge. The voltage across the capacitor decreases exponentially from the
initial value U0 to zero. As the capacitance or resistance increases, the time of the
capacitor discharge increases according to Eq. 2.7.
Professor V. S. Markin developed a mathematical treatment of a capacitor
discharge in passive electrical circuits in plants (Volkov et al. 2010c). It was
observed that capacitor discharge through the Mimosa pudica petiole has a two-
exponential character (Volkov et al. 2010c). Therefore, the electrical circuit can be
modeled as it is shown in Fig. 2.8b. In our experiment the capacitor C1 was
charged to voltage U0 and the circuit was closed.
After closing the circuit, the electrical potentials U1 and U2 at capacitors C1 and
C2 depend on time according to the equations:
8
< C1 dU1 ¼  1 ðU1  U2 Þ
dt R1   ð2:9Þ
: C2 dU2 ¼  1 U1  1 þ 1 U2
dt R1 R1 R2

with initial conditions

U1 ½0 ¼ U0 ; U2 ½0 ¼ 0 ð2:10Þ
2 Plant Electrostimulation and Data Acquisition 57

It is convenient to introduce parameters of time:

h 1 ¼ R1 C 1 ; h2 ¼ R2 C 2 ; h 3 ¼ R2 C 1 ð2:11Þ
Voltage U2 can be excluded from the system of Eq. 2.9 giving the single
equation for U1:

d2 U 1 dU1
h1 h2 þ ðh1 þ h2 þ h3 Þ þ U1 ¼ 0 ð2:12Þ
dt2 dt
Solving this equation one can find the time course of U1:
(2 3
6 h1 þ h2  h3 7
U 1 ðt Þ ¼ U 0 4qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi þ 15
2
4h1 h2 þ ðh1 þ h2 þ h3 Þ
2  qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi3
t h1 þ h2 þ h3 þ 4h1 h2 þ ðh1 þ h2 þ h3 Þ2
6 7
 Exp6
4
7
5
2h1 h2
2 3
6 h1  h2 þ h3 7
þ 4qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi þ 15
2
4h1 h2 þ ðh1 þ h2 þ h3 Þ
2  qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi39
t h1 þ h2 þ h3  4h1 h2 þ ðh1 þ h2 þ h3 Þ2 > >
6 7=
6
 Exp4 7
2h1 h2 5>
>
;

ð2:13Þ
If the experimental dependence can be approximated with function
st st
UðtÞ ¼ A1 e 1 þ A2 e 2 ð2:14Þ
and parameters A1, A2, s1, and s2 can be determined from the experiment, then one
can evaluate the elements of the equivalent circuit in Fig. 2.8b.
To find parameters h1, h2, h3 simultaneously, Markin solved the set of nonlinear
equations derived from comparison of Eqs. 2.13 and 2.14:
2 3
6 h1 þ h2  h3 7
A1 ¼ U0 4qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi þ 15
2
4h1 h2 þ ðh1 þ h2 þ h3 Þ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1 h 1 þ h 2 þ h 3 þ 4h1 h2 þ ðh1 þ h2 þ h3 Þ2
¼
s1 2h1 h2
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
1 h1 þ h2 þ h3  4h1 h2 þ ðh1 þ h2 þ h3 Þ
¼ ð2:15Þ
s2 2h1 h2
58 E. Jovanov and A. G. Volkov

(a) (b)

U0 R2 C2
R2 C1 C1 U0
R1

(c)

R2 C1 U0
D1 D2 C2
R1

Fig. 2.8 Electrical equivalent schemes of a capacitor discharge in a plant tissue. Abbreviations:
C1 charged capacitor from voltage source U0; C2 capacitance of plant tissue; R resistance in plant
tissue; D diode as a model of a voltage-gated channel

Finding from here h1, h2, and h3 and using the set of Eq. 2.11, one can deter-
mine the value of elements C2, R1, R2 in the equivalent circuit shown in Fig. 2.8b.
Volkov et al. (2010c) estimated these parameters in the M. pudica petiole.
Another interesting parameter is input resistance that can be defined as
U1
Rinput ¼  ð2:16Þ
C1 ddUt1

This parameter is also often analyzed in electrical impedance spectroscopy

studies of biological tissues (Laarabi et al. 2005; Zhang and Willison 1991; Wang
et al. 1994). However, there is a problem with interpretation of electrical
impedance method, because a few different equivalent circuit models can have
identical impedances (McAdams and Jossinet 1996).

2.3.3 Patch Clamp and Electrochemical Impedance Methods

The most frequently used methods for the evaluation of electrical circuits in plants are
patch clamps, electrochemical impedance measurement, and electric charge stimu-
lation. The patch clamp method can be used to study electrical characteristics of
individual ionic channels in a biological membrane in vitro. Pipette-based recording of
membrane currents is the mainstay in the characterization of cellular ion channels.
Traditional patch clamp recording is accomplished by using a micromanipulator to
position the tip of a glass pipette against the membrane of a cell. In voltage clamp
mode, the membrane is clamped to a preset potential, and the current required main-
taining this potential is recorded. Current recordings with different electrical protocols
and in the presence of different reagents are used to characterize ion channel properties.
The electrochemical impedance method measures static electrical parameters, such
as resistance and capacitance, at high-frequency alternative currents (AC). However,
different electrochemical circuits can have the same electrochemical impedance.
2 Plant Electrostimulation and Data Acquisition 59

The description of equivalent electrical circuits based on electrochemical impedance

AC measurements is based on the researcher’s intuition and can lead to various
mistakes (McAdams and Jossinet 1996). Moreover, this method cannot characterize
dynamic changes and nonlinear events, such as ion channel opening and closing.

2.4 Plant Electrostimulation

2.4.1 Plant Movements Induced by DC Electrostimulation

Mechanical movements in M. pudica were induced by very high applied voltages

(Balmer and Franks 1975; Bose 1918; Gardiner 1888; Jonas 1970; Ritter 1811).
Balmer and Franks (1975) briefly applied 200–400 V between the soil and the
primary pulvinus to measure the contractile characteristics of a petiole. They
estimated that the threshold voltage was about 25 V with any electrode polarity.
Jonas (1970) used a 0.5 lF capacitor charged by 50, 100, and 150 V for electr-
ostimulation and found that there were oscillations of leaves and fast petiolar
movement after the application of an electrical shock. When Yao et al. (2008)
applied 9 V to M. pudica, the petioles bent downward and the pinnae closed.
Volkov et al. (Volkov et al. 2010e) investigated the mechanical movements of the
pinnae and petioles in M. pudica induced by the electrical stimulation of a pul-
vinus, petiole, secondary pulvinus, or pinna by low electrical voltage and charge
(Fig. 2.9). The threshold value was 1.3–1.5 V of applied voltage and 2–10 lC
charge for the closing of the pinnules. Both voltage and electrical charge are
responsible for the electrostimulated closing of a leaf (Volkov et al. 2010a, b, c, d).
The electrical stimulus between a midrib and a lobe closes the Venus flytrap
leaf by activating motor cells without mechanical stimulation of trigger hairs
(Markin et al. 2008; Volkov et al. 2007, 2008a, b, 2009a, b). The closing time of
the Venus flytrap by electrical stimulation is 0.3 s, the same as mechanically
induced closing. The Venus flytrap can accumulate small subthreshold charges,
and when this threshold value is reached, the trap closes. Ion channel blockers such
as Ba2+ and TEACl as well as uncouplers such as FCCP, 2,4-dinitrophenol and
pentachlorophenol dramatically decrease the speed of the trap closing (Volkov
et al. 2008c). Electrical stimulation can be used to study mechanisms of fast
activity in motor cells of the plant kingdom.

2.4.2 DC Electrostimulation of Plants Can Induce Gene

Expression, Enzymatic Systems Activation, Electrical
Signaling, and Influences on Plant Growing

Excitability is a fundamental property that plants exhibit at the whole plant, tissue,
and cellular levels. This property allows the cells, tissues, and organs of plants to
60 E. Jovanov and A. G. Volkov

LIGHT

FARADAY
CAGE NI PXI-1042Q Computer
NI PXI-4071 Data Acquisition
Board
Petiole NI PXI-4110 DC Power Supply

Ag/AgCl
C1 47 F

Ag/AgCl

DPDT switch
Pulvinus

Fig. 2.9 Experimental setup for plant electrostimulation using the charge stimulation method
and electrical signal measurements. We used PXI (PCI eXtensions for Instrumentation), a rugged
PC-based platform, as a high-performance measurement and automation system (National
Instruments, Texas)

operate in concert to adapt its internal conditions and external reactions in

response to environmental stimulants referred to as irritants. The excitation waves,
or action potentials, in higher plants are thought to be the mechanism behind
intercellular and intracellular communication. Action potentials are signals caused
by the depolarization of cellular membranes. Mechanical, physical, or chemical
irritants affect not only the location of occurrence, but these irritants may also
affect the entire plant as well. In plant species, the velocity of electrical signals
depends on many factors, including the intensity of the irritation, temperature,
chemical treatment, or mechanical wounding. The excitation reaction may travel
between the top of the stem and the root in either direction.
Plant electrostimulation can have influence on the growth of plants (Takamura
2006). Mizuguchi et al. (1994) applied 1 V to the cultured solution and it accel-
erated by 30% the growth of bean sprouts. Goldsworthy (2006) described in his
review how natural and artificial electrical fields stimulate plant growth in the
electroculture experiments. Electrical fields can activate growth-promoting genes
(Goldsworthy 2006). Electroculture experiments show that electrical fields from
the aurora borealis are responsible for green and healthy vegetation in Arctic
(Lemström 1904). Goldsworthy (2006) suggested that ‘‘plants seem to be using the
very strong electrostatic fields associated with thunderstorms as signal to let them
make the best use of the rain.’’
2 Plant Electrostimulation and Data Acquisition 61

Fromm and Spanswick (1993) measured action potentials induced by electr-

ostimulation of willow shoots. The threshold value of electrical stimulation was
function of duration of electrical stimuli (6 V during 1 s, 3–4 V during 2 s or
10 nA in 1 s, 3 nA in 2 s, 2 nA during 4 s). Such dependence on time shows that
both voltage and electrical charge are important for the plant electrical response.
We can estimate the threshold charge of 6–10 nC by multiplying the threshold
current by time of polarization. Amplitude of action potentials was 30–50 mV
with velocity of 2 cm/s.
Inaba et al. (1995) applied DC current of 1–3 mA to cucumber (Cucumis
sativus L.) and found induction of ethylene synthesis and activation of 1-amino-
cyclopropane-1-carboxylic acid synthase. Inaba et al. (1995) also evaluated
parameters of the equivalent electrical circuit of plant tissues.
Herde et al. (1995, 1996) found that electrical current application (10 V, 30 s)
activated pin2 gene expression in tomato plants and increases endogenous level of
abscisic acid.
Stanković and Davies (1996, 1997) demonstrated that electrical stimulation
of tomato plants by 9 V during 3–4 s occasionally induces ‘‘genuine’’ action
potentials with amplitude of 40 mV and speed of 3.5–4.5 mm/s and elicits
systemic pin2 gene expression. Propagation of electrical signals in response to
electrostimulation was found in 20% of tomato plants only (Stankovic and
Davies 1996, 1997). Authors did not analyze the threshold level of a stimu-
lation voltage and possible dependence of action potential on amplitude and
polarity of applied voltage. Stankovic and Davies (1996, 1997) found after 9 V
electrostimulation that ‘‘5-fold or greater increase in pin2 mRNA levels occurs
within 1 h’’.
Mishra et al. (2001) studied action potential propagations with velocity of
270 m/s and a latency of 400 ls from root to shoot in Sorghum bicolor evoked
by electrostimulation. The threshold current was 100 lA during 0.3 ms. These
values correspond to the threshold charge of 30 nC. In 7-day-old seedling the
threshold charge was 0.45 lC. According to the strength-duration curve of
S. bicolor seedling the threshold charge varies from 30 to 3 lC (Mishra et al.
2001).
Dziubinska et al. (2001) applied electrical stimuli of 1–2 V during 5 s to the
basal part of the stem of Helianthus annuus and recorded propagation of action
potentials along the stem with an amplitude of 42 mV with speed of 0.18 cm/s.
Authors were unable to evoke action potentials by electrical stimulation of leaves
(Dziubinska et al. 2001).
Krol et al. (2006) triggered action potentials in the Venus flytrap by electrical
stimuli up to 4 V.
Favre and Agosti (2007) described voltage-dependent ‘‘action potentials’’ in
Arabidopsis thaliana induced by 3–18 V pulses. Amplitude of these ‘‘action
potentials’’ was between 10 and 80 mV with an absolute refractory period of
20 min and a speed of propagation of 0.8–1.4 mm/s. Amplitude and velocity of
electrical responses was a function of applied voltage.
62 E. Jovanov and A. G. Volkov

Fig. 2.10 Time dependence of electrical discharge in M. pudica pulvinus between electrodes
located along the pulvinus and connected to 47 lF charged capacitor (a, c) Time dependence of
electrical discharge in the M. pudica’s pulvinus between electrodes located along the pulvinus
and connected to a charged capacitor in logarithmic coordinates (b, d)

2.4.3 Anisotropy and Nonlinear Properties of Biologically

Closed Electrochemical Circuits in Plants

Electrical circuits in Fig. 2.8a, b are not sensitive to the polarity of applied voltage.
We found that after threshold value of electrostimulating potential there is a strong
deviation in logarithmic coordinates from Eq. 2.8 predictions and plant electrical
responses depend on polarity of applied voltage (Fig. 2.10). The deviation of a
capacitor discharge from a linear dependence in logarithmic coordinates can be
described by the equivalent electrical schemes shown in Fig. 2.8b, c (Volkov et al.
2009b, 2010a b, c, d). If the capacitor discharge is represented by two-exponential
functions and does not depend on polarity of electrodes in the plant tissue, the
deviation from linear dependence can be described by Fig. 2.8b. If the response
changes with the polarity of stimulation, a rectifier-based model represented in
Fig. 2.8c must be used. Kinetics of a capacitor discharge depends on the polarity
of electrodes in the Aloe vera leaf, Venus flytrap, and M. pudica (Figs. 2.10 and
2.11). Figure 2.11 shows the difference in the kinetics of a capacitor discharge as a
function of the polarity of stimulation as represented in Fig. 2.10a, c. Dependence
of a capacitor discharge on the polarity of electrodes in the M. pudica leaf, shown
2 Plant Electrostimulation and Data Acquisition 63

Fig. 2.11 Time dependence 0.02

of voltage differences
(Fig. 2.10a ? c) during
0.00 ±0.05 V
electrical discharge in the
M. pudica’s pulvinus between
electrodes of different -0.02
±1.5 V

ΔU / V
polarities located along the
M. pudica pulvinus and
-0.04
connected to 47 lF charged
capacitor
-0.06

-0.08
0 20 40 60 80 100
Time / s

in Figs. 2.10 and 2.11, can be explained by a change in resistivity with applied
potential due to opening of ion channels, which can be modeled by diodes in
Fig. 2.8c. Opening of voltage-gated channels induce the effect of electrical
rectification shown in Fig. 2.11. We found similar rectification effects in the Venus
flytrap (Volkov et al. 2009b), A. vera (Volkov et al. 2011a, b), and M. pudica
(Volkov et al. 2010a, b, c, d, e, 2011a). We modeled voltage-gated channels using
silicon rectifier diode and reproduced experimental dependencies of a capacitor
discharge in plant tissue (Volkov et al. 2009b).

2.4.4 Circadian Variations in Biologically Closed

Electrochemical Circuit in Plants

The circadian clock regulates a wide range of electrophysiological and develop-

mental processes in plants. The circadian clock is an endogenous oscillator with a
period of approximately 24 h; its rhythm is linked to the light–dark cycle.
Molecular mechanism underlying circadian clock function is poorly understood,
although it is widely accepted for both plants and animals that are based on
circadian oscillators. The circadian clock in plants is sensitive to light, which
resets the phase of the rhythm. The circadian clock was discovered by De Mairan
(1729) in his first attempt to resolve experimentally the origin of rhythm in the leaf
movements of M. pudica. This rhythm continued even when M. pudica was
maintained under continuous darkness. M. pudica is a nyctinastic plant that closes
its leaves in the evening; the pinnules fold together and the whole leaf droops
downward temporarily until sunrise. The leaves open in the morning due to a
circadian rhythm, which is regulated by a biological clock with a cycle of about
24 h. During photonastic movement in M. pudica, leaves recover their daytime
position. During a scotonastic period, the primary pulvini straighten up and pairs
of pinnules fold together about the tertiary pulvini. The closing of pinnae depends
upon the presence of phytochrome in the far-red absorbing form.
64 E. Jovanov and A. G. Volkov

Volkov et al. (2011a,c) found, for the first time, the direct influence of a
circadian clock on biologically closed electrochemical circuits in vivo using
the Charge Stimulating Method. The electrostimulation of a sensitive plant
M. pudica and A. vera was provided with different timing and different
voltages. A. vera (L.) is a member of the Asphodelaceae (Liliaceae) family
with crassulacean acid metabolism (CAM). In A. vera, stomata are open at
night and closed during the day. CO2 acquired by A. vera at night is tempo-
rarily stored as malic and other organic acids, and is decarboxylated the
following day to provide CO2 for fixation in the Benson-Calvin cycle behind
closed stomata.
A. vera is a model for the study of plant electrophysiology with crassulacean
acid metabolism.
Resistance between Ag/AgCl electrodes in the leaf of A. vera was higher during
the day than at night. Discharge of the capacitor in A. vera at night was faster than
during the day. Discharge of the capacitor in a pulvinus of M. pudica was faster
during the day. The biologically closed electrical circuits with voltage-gated ion
channels in M. pudica are also activated the next day, even in the darkness. These
results show that the circadian clock can be maintained endogenously and has
electrochemical oscillators, which can activate ion channels in biologically closed
electrochemical circuits. Initial difference in the speed of the capacitor discharge
(faster during the day), can be explained by activation of ion channels, equivalent
to the high rectification effect. This effect depends on the applied stimulation
voltage.
Isolated pulvinar protoplasts are responsive to light signals in vitro (Coté 1995;
Kim et al. 1992, 1993). In the dark period, the closed inward-directed K+ channels
of extensor cells are opened within 3 min by blue light. Conversely, the inward-
directed K+ channels of flexor cells, which are open in the darkness, are closed by
blue light. In the light period, however, the situation is more complex. Premature
darkness alone is sufficient to close the open channels of extensor protoplasts, but
both darkness and a preceding pulse of red light are required to open the closed
channels in the flexor protoplasts (Kim et al. 1992, 1993).
The biologically closed electrical circuits with voltage-gated ion channels in
M. pudica are activated the next day even in the darkness. This phenomenon can be
caused by biological clock in M. pudica. The nonlinear effect of activation of elec-
trical circuits during the daytime is stronger than during the next day in the darkness.
In contrast to A. vera the discharge of the capacitor in a pulvinus of M. pudica
was faster during the day, because A. vera is a CAM plant.
Our results (Volkov et al. 2011a, c) demonstrate that the circadian clock can be
maintained endogenously, probably involving electrochemical oscillators, which
can activate or deactivate ion channels in biologically closed electrochemical
circuits. This circadian rhythm can be related to the differences found in the
membrane potentials during the day- and nighttime, which were found in different
plants (Kim et al. 1992; 1993; Racusen and Satter 1975; Scott and Gulline 1975;
Thomas and Vince-Prue 1997). The expression of many ion transporters in plants
is regulated by the circadian rhythm (Lebaudy et al. 2008).
2 Plant Electrostimulation and Data Acquisition 65

2.5 Conclusion

The information gained from plant electrostimulation can be used to elucidate the
intracellular and intercellular communication in the form of electrical signals
within plants. Monitoring the electrical signaling in higher plants represents a
promising method to investigate electrical communication during environmental
changes.

Acknowledgments This work was supported by the grant W911NF-11-1-0132 from the U.S.
Army Research Office.

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